Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass SpectrometryEloy R. Wouters,1 Pan Mao,2 Jean-Jacques Dunyach,1 Daojing Wang2 1Thermo Fisher Scientific, San Jose, CA, USA; 2Newomics Inc., Emeryville, CA, USA

2 Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Results Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays To overcome the limitations found in prior multisprayer designs, such as the chemical and thermal stability of the material [3], we have developed novel monolithic, silicon-chip-based, multinozzle emitter arrays [4].

These devices are produced using photolithography and deep reactive ion etching from two silicon wafers that are bonded together to form an internal channel (Figure 1). All surfaces that surround the channel are wet oxidized to form a thick oxide layer, and the resulting protruding nozzles have a square cross section profile (Figure 2a-c) and constituted of SiO2. The standard silicon microchip fabrication technology ensures reproducibility between devices as well as the potential of creating of a large number of small high aspect ratio structures that allow for the creation of a local electric field strength strong enough to form a Taylor cone.

The multinozzle emitter array chips also offer a straightforward solution to the problem of splitting the liquid flow without leaks or dead volume, which could prevent their practical use. The internal channel of the chip is 20 mm long, 100 μm wide, and has a depth of 10 μm (volume is 20 nL), and can in the future be packed with resin beads to perform on-chip LC separations.

The issue of the poor geometric overlap of a linear array of emitters with the circular atmospheric inlet to a mass spectrometer, as we described in Ref. 4, has been addressed by employing a rectangular slotted ion transfer tube that was perfectly aligned with the linear emitter array.

Table 1 lists the device geometry design parameters that have been varied during the microchip production process: emitter tip spacing, protrusion, emitter channel size, and number of emitters ranging from 1 to 80. A small selection of these microchip emitter arrays has so far been experimentally investigated.

Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Eloy R. Wouters1, Pan Mao2, Jean-Jacques Dunyach1, Daojing Wang2 1Thermo Fisher Scientific, San Jose, CA, 2Newomics Inc., Emeryville, CA

Conclusion Multinozzle emitter arrays were fabricated from a monolithic silicon substrate,

creating sufficient electric field strength to form and sustain a stable uniform array of Taylor cones without coalescence of the fluid as well as reproducible sensitivity between emitter chips

Linear geometry of the multinozzle emitter array makes feasible the optimal geometric overlap with the slotted inlet on the TSQ QuantivaTM triple quadrupole mass spectrometer

Possible solution for microflow electrospray applications with reduced ion suppression

Intensity gains observed in infusion

Further experimentation is ongoing: to find the optimal nozzle cross section for each number of nozzles to evaluate the potential for increased linearity of the ion source and more

uniform ionization efficiency for different analytes to establish overall ruggedness of the device for LC-MS-MS applications

with samples in biological matrices which is a key parameter for its success

Future directions include the stacking of several arrays to increase the number of nozzle per emitter while retaining the geometric overlap with the slotted atmospheric pressure inlet.

References 1. A.J. Rulison and R.C. Flagan, Scale-up of Electrospray Atomization Using Linear

Arrays of Taylor Cones, Rev. Sci. Instrum. 64, 683 (1993)

2. J.F. de la Mora and I.G. Loscertales, The Current Emitted by Highly Conducting Taylor Cones, J. Fluid Mech. 260, 155 (1994)

3. K. Tang, Y. Lin, D.W. Matson, T. Kim, and R.D. Smith, Generation of Multiple Electrosprays Using Microfabricated Emitters Arrays for Improved Mass Spectrometric Sensitivity, Anal. Chem. 73, 1658 (2001)

4. P. Mao, H-T. Wang, P. Yang, and D. Wang, Multinozzle Emitter Arrays for Nanoelectrospray Mass Spectrometry, Anal. Chem. 83, 6082 (2011)

5. B.Q.T. Si, D. Byun, and S. Lee, Experimental and Theoretical Study of a Cone-jet for an Electrospray Microthruster Considering the Interference Effect in an Array of Nozzles, Aerosol Science 38, 924 (2007)

Acknowledgements Financial support from NIH SBIR program to Newomics Inc. is gratefully acknowledged. We thank the UC Berkeley Marvell Nanofabrication Laboratory for facility access.

Overview Purpose: Evaluation of linear multinozzle emitter arrays for low flow mass spectroscopy.

Methods: Infusion at microflow rates and LC-MS-MS at nanoflow rates on a triple quadrupole mass spectrometer equipped with a slotted tube.

Results: Successful generation of a large number of Taylor cones and stable ion signal over a wide range of liquid flow rates.

Introduction The soft ionization provided by nano-electrospray sources used in mass spectrometry is an important tool for qualitative and quantitative measurements of peptides and proteins. The electrospray ionization efficiency is typically improved at nanospray flow rates due to the smaller size of the original droplets formed at the emitter tip.

However, single capillary-based nanospray lacks the speed and robustness that is required for many clinical and bio-pharmaceutical applications. One approach is to maintain microflow LC conditions while achieving nanoflow sensitivity by dividing the eluent from the LC system over a multiple of electrospray emitters [1], each operating at a fraction of the total liquid flow rate. The theoretical ion current increase is proportional to the square root of the number of emitters [2].

Methods Various samples were used to evaluate the multinozzle emitter arrays, provided by Newomics Inc. with exploratory infusion experiments at flow rates between 600 nL/min and 10 μL/min. The samples were chosen to cover several classes of compounds (small drug molecules, various proteins and peptides). Under standard nanospray conditions, these samples form singly as well as multiply charged protonated molecular ions, and were dissolved in solvent with different acidities to investigate the ionization process for the new multinozzle emitter arrays.

• “cal-mix”, a mixture of 2.0 ng/μL Caffeine, 5.5 pmol/μL MRFA (Met-Arg-Phe-Ala), and Ultramark 1621, dissolved in 1:1:2 methanol:water:acetonitrile (1.0% Acetic Acid)

• 1.0 pmol/μL Angiotensin I dissolved in 1:1 methanol:water (0.1% Acetic Acid) • 1.0 pmol/μL Hemoglobin A dissolved in 1:1 acetonitrile:water (0.2% Formic Acid)

A separate assay, including constituents of Bovine Serum Albumin (BSA) and Enolase diluted in E. Coli digest was used to characterize sensitivity at a nano-flow rate (300 nL/min). Six target constituents were resolved using a Thermo ScientificTM AcclaimTM PepMapTM RSLC 50μm x 15cm, C18, 2μm, 100 Å column over a 25 min duration gradient program controlled by a Thermo ScientificTM EASY-nLC™ 1000 system. Composition of the mobile phases was varied in multiple steps from 5% aqueous (0.1% Formic Acid) to 90% methanol (0.1% Formic Acid)

The assays were analyzed on the Thermo ScientificTM TSQ QuantivaTM triple-stage quadrupole mass spectrometer equipped with a slotted ion transfer tube held at 270 – 300 °C. The mass spectrometer was operated in the full scan mode at the 1000 Da/sec scan rate, or alternatively in the SRM mode with a 10 msec cycle time and the Argon collision gas pressure was 1.5 mTorr.

The multinozzle emitter array device was tightly clamped between a PEEK plate and a steel plate, where the PEEK plate has a threaded hole to accept standard fittings for capillary tubing and a gasket on the device side to prevent fluid leakage. The steel plate is connected to the HV power supply of the mass spectrometer (Figures 2d). This sandwiched assembly was mounted on a translation stage in front of the mass spectrometer inlet using a modified Thermo ScientificTM Nanospray Flex™ ion source (Figure 2e).

Establishing Taylor Cones on Multinozzle Emitter Arrays Experimental conditions were optimized to create a sufficiently high local electric field on the prototype multinozzle (n=1-100) emitter arrays to successfully form and sustain a uniform array of Taylor cones for a wide range of samples and flow rates without coalescence of the liquid as observed with an optical microscope (Figure 3). Note that at lower per nozzle flow rates and at smaller nozzle cross sections it was difficult to illuminate and observe the finer mist, hence the photographs presented here are at the high end of the flow rate range that was studied.

It can be observed in Figure 3 that the Taylor cones of the nozzles remained parallel with a remarkably small deviation at either end. Further, this deviation angle was found to increase with the liquid flow rate.

It is worth emphasizing that our emitter arrays allow for the stable operation at extremely high flow rates before eventually suffering the post nozzle coalescence often reported with alternative designs from others at much lower flow rates. The arrays with the larger 10 × 10 μm nozzles can be operated without backpressure limitations at flow rates that far exceed the normal nanoflow regime, while the individual nozzles still experience a flow rate well within or even on the low end of this range. For example, in Figure 4c, 15 μl/min was consumed with a low per nozzle flow rate of 300 nL/min. Potential applications are envisioned of the multinozzle emitter arrays for assays at microspray flow rates with reduced ion suppression.

The distance between the emitter array chip and the mass spectrometer inlet as well as the spray voltage were optimized in each experiment. It was found that compared to an uncoated pulled tip emitter (operated typically at 1.5 kV), a higher voltage was needed both for the onset of Taylor cones, as well as for optimal signal, with optimized values between 3.3 and 3.6 kV. The voltage needed to be increased up to the breakdown limit, a likely indication that for those number of emitters in the array, and under those flow conditions, the local electric field is close to being sufficient. A possible explanation would be the interference effects by the electric fields formed by neighboring emitters [5], and the current design perhaps could benefit from the inclusion of an extraction electrode to enhance the shielding between neighboring nozzles.

Optimum signal was found for those emitter arrays with overall widths that were not exceeding that of the slotted inlet of the mass spectrometer (i.e. 2.0 mm).

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

PO64083-EN 0614S

FIGURE 4. Total Ion Chromatograms (TIC) showing the stability of the signal in top panels (a)–(c) and, in bottom panels (a)–(c), the time integrated mass resolved spectra (Q1MS mode at 0.7 Da resolution) of Hemoglobin A (1.0 pmol/μL infusion) employing multinozzle emitter chips of 10 × 10 μm each: (a) 25-nozzle array at 3.1 kV at 600 nL/min (24 nL/min per nozzle); (b) 50-nozzle array at 3.6 kV at 600 nL/min (12 nL/min per nozzle); (c) 50-nozzle array at 3.4 kV at 2 μL/min (40 nL/min per nozzle). The base peak intensity as a function of total liquid flow rate is depicted for several emitter arrays (d).

Infusion at Nano and Micro Flow Rates The emitter array was found to produce short and long term stable peak intensities in the mass spectrometer (Figure 4).

It can also be observed in Figures 4a – 4c that the charge state distribution was not dependent number of nozzles or liquid flow rate. Additional studies (not shown) also established that it was independent of other device design parameters, and similar to that of a standard nanospray emitter.

Comparing the signal intensity for a fixed total liquid flow rate between emitter arrays with different number of nozzles with the same 10 × 10 μm cross section (Figure 4d), it can be seen that the emitter arrays show increased signal relative to a standard single uncoated pulled tip nanospray emitter (10 μm diameter).

The advantages provided by the photolithographic reproduction on chip-to-chip reproducibility can be judged by the similarity of signal at various flow rates of three 25-nozzle emitter arrays in Figure 4d.

Nanoflow LC-MS-MS Experiments A nanoflow chromatography SRM experiment was performed for the 25-nozzle (10 × 10 μm) emitter array by targeting six constituents of a mixture of BSA and Enolase in an E. Coli digest: TFAEALR (m/z 404.22), AEFVEVTK (m/z 461.75), IGSEVYHNLK (m/z 580.31), LVNELTEFAK (m/z 582.32), NVNDVIAPAFVK (m/z 643.86), and HLVDEPQNLIK (m/z 653.36). We found that the average peak intensities showed good reproducibility between replicate injections (Figure 5). At the concentrations studied this assay is not signal to noise limited and should well represent the number of ions generated.

The slight peak tailing observed in the chromatograms is indicative of post column dead volume, and we identified places to improve the interface to the LC. We are currently optimizing the connections between the multinozzle emitter arrays and LC column to minimize this dead volume.

Further experimentation is underway to compare emitter arrays with different number of nozzles with varying cross sections. For example, for total flow of 300 nL/min, the flow through one of the nozzles in a 50 emitter array is only 6 nL/min, and a 10 × 10 μm nozzle cross section geometry might be less than optimal. Further experiments with a smaller nozzle for the emitter arrays with larger number of nozzles are required.

Parameter Values Number of nozzles 1, 5, 10, 20, 40, 80 Total array width 1.0, 1.75, 2.0, 3.5 mm Nozzle cross section 3 × 3, 5 × 5, 10 × 10 μm Protrusion of nozzle above wafer ~100 μm Inter nozzle spacing 30, 35, 40, 60, 80, 100, 150, 200 μm

TABLE 1. Design parameters of the silicon-chip-based multinozzle emitter arrays. FIGURE 1. Cross section of the monolithic multinozzle emitter array chip. The chip was produced using standard silicon microchip fabrication techniques. Two wafers were bonded together to form an internal channel. In published experiments, this channel could be packed with different kinds (C4 and C18) of beads for on-chip LC separation.

FIGURE 3. Optical microscope images of separate Taylor cones formed by (a) 10-nozzle emitter array (10 × 10 μm nozzles) at a spray voltage of 2.8 kV and 3 μL/min infusion of Angiotensin I, i.e. a flow rate through each nozzle of 300 nL/min; (b) 25-nozzle emitter array (10 × 10 μm nozzles) at 3.5 kV spraying 2 μL/min of Hemoglobin A, i.e. a flow rate through each nozzle of 80 nL/min; (c) 50-nozzle emitter array (10 × 10 μm nozzles) at 4.2 kV spraying 15 μL/min of “cal-mix”, i.e. a flow rate through each nozzle of 300 nL/min; (d) 50-nozzle emitter array (3 × 3 μm nozzles) at 3.8 kV spraying 5 μL/min of acetonitrile (0.1% Formic Acid): 100 nL/min per nozzle. The area represented in the micrographs measures 3.0 × 2.0 mm.

(c)

(a)

2.0

mm

wid

e in

let

FIGURE 2. Scanning electron microscope images of sharpened-end multinozzle emitters: (a) 1-nozzle emitter with a cross section of 10 × 10 μm. (b) 20-nozzle emitter with a cross section of 5 x 5 μm. (c) 40-nozzle emitter with a cross section of 2 × 2.5 μm. The zoom-out and close-up views of each emitter are shown in the subpanels (i) and (ii), respectively. Photographs of (d) the multinozzle emitter mounted in a prototype holder, and (e) the holder mounted on the Nanospray Flex NG ion source in front of the slotted ion transfer tube inlet to the mass spectrometer.

d

e

a

b

c

FIGURE 5. Total Ion Chromatogram (TIC) showing the elution of six constituents from a mixture of BSA and Enolase at a flow rate of 300 nL/min. The bar charts on the right represent the peak area for three replicate on column injections of 10 femtomole using a 25-nozzle emitter array of 10 × 10 μm nozzles at 3.3 kV.

0.E+00

1.E+05

NVNDVIAPAFVK 0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05 LVNELTEFAK

0.0E+00

5.0E+04

1.0E+05 HLVDEPQNLIK

0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05 AEFVEVTK

NL: 3.73E4 TIC MS

IGSE

VYH

NLK

AEF

VEVT

K

HLV

DEP

QN

LIK

TFAE

ALR

LVN

ELTE

FAK

N

VND

VIAP

AFVK

BSA-Enolase-10femtomole-newomics-25-run2

13 14 15 16 17 18 19 20 21 Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

(d)

(b) (a)

(c)

(b)

0.0E+00

5.0E+06

1.0E+07

1.5E+07

0 0.5 1 1.5 2 2.5

Bas

e pe

ak in

tens

ity

Total flow rate (μL/min)

25-emitter #1 25-emitter #2 25-emitter #3

50-emitter single pulled tip

(d)

3Thermo Scientific Poster Note • PN-64083-ASMS-EN-0614S

Results Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays To overcome the limitations found in prior multisprayer designs, such as the chemical and thermal stability of the material [3], we have developed novel monolithic, silicon-chip-based, multinozzle emitter arrays [4].

These devices are produced using photolithography and deep reactive ion etching from two silicon wafers that are bonded together to form an internal channel (Figure 1). All surfaces that surround the channel are wet oxidized to form a thick oxide layer, and the resulting protruding nozzles have a square cross section profile (Figure 2a-c) and constituted of SiO2. The standard silicon microchip fabrication technology ensures reproducibility between devices as well as the potential of creating of a large number of small high aspect ratio structures that allow for the creation of a local electric field strength strong enough to form a Taylor cone.

The multinozzle emitter array chips also offer a straightforward solution to the problem of splitting the liquid flow without leaks or dead volume, which could prevent their practical use. The internal channel of the chip is 20 mm long, 100 μm wide, and has a depth of 10 μm (volume is 20 nL), and can in the future be packed with resin beads to perform on-chip LC separations.

The issue of the poor geometric overlap of a linear array of emitters with the circular atmospheric inlet to a mass spectrometer, as we described in Ref. 4, has been addressed by employing a rectangular slotted ion transfer tube that was perfectly aligned with the linear emitter array.

Table 1 lists the device geometry design parameters that have been varied during the microchip production process: emitter tip spacing, protrusion, emitter channel size, and number of emitters ranging from 1 to 80. A small selection of these microchip emitter arrays has so far been experimentally investigated.

Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Eloy R. Wouters1, Pan Mao2, Jean-Jacques Dunyach1, Daojing Wang2 1Thermo Fisher Scientific, San Jose, CA, 2Newomics Inc., Emeryville, CA

Conclusion Multinozzle emitter arrays were fabricated from a monolithic silicon substrate,

creating sufficient electric field strength to form and sustain a stable uniform array of Taylor cones without coalescence of the fluid as well as reproducible sensitivity between emitter chips

Linear geometry of the multinozzle emitter array makes feasible the optimal geometric overlap with the slotted inlet on the TSQ QuantivaTM triple quadrupole mass spectrometer

Possible solution for microflow electrospray applications with reduced ion suppression

Intensity gains observed in infusion

Further experimentation is ongoing: to find the optimal nozzle cross section for each number of nozzles to evaluate the potential for increased linearity of the ion source and more

uniform ionization efficiency for different analytes to establish overall ruggedness of the device for LC-MS-MS applications

with samples in biological matrices which is a key parameter for its success

Future directions include the stacking of several arrays to increase the number of nozzle per emitter while retaining the geometric overlap with the slotted atmospheric pressure inlet.

References 1. A.J. Rulison and R.C. Flagan, Scale-up of Electrospray Atomization Using Linear

Arrays of Taylor Cones, Rev. Sci. Instrum. 64, 683 (1993)

2. J.F. de la Mora and I.G. Loscertales, The Current Emitted by Highly Conducting Taylor Cones, J. Fluid Mech. 260, 155 (1994)

3. K. Tang, Y. Lin, D.W. Matson, T. Kim, and R.D. Smith, Generation of Multiple Electrosprays Using Microfabricated Emitters Arrays for Improved Mass Spectrometric Sensitivity, Anal. Chem. 73, 1658 (2001)

4. P. Mao, H-T. Wang, P. Yang, and D. Wang, Multinozzle Emitter Arrays for Nanoelectrospray Mass Spectrometry, Anal. Chem. 83, 6082 (2011)

5. B.Q.T. Si, D. Byun, and S. Lee, Experimental and Theoretical Study of a Cone-jet for an Electrospray Microthruster Considering the Interference Effect in an Array of Nozzles, Aerosol Science 38, 924 (2007)

Acknowledgements Financial support from NIH SBIR program to Newomics Inc. is gratefully acknowledged. We thank the UC Berkeley Marvell Nanofabrication Laboratory for facility access.

Overview Purpose: Evaluation of linear multinozzle emitter arrays for low flow mass spectroscopy.

Methods: Infusion at microflow rates and LC-MS-MS at nanoflow rates on a triple quadrupole mass spectrometer equipped with a slotted tube.

Results: Successful generation of a large number of Taylor cones and stable ion signal over a wide range of liquid flow rates.

Introduction The soft ionization provided by nano-electrospray sources used in mass spectrometry is an important tool for qualitative and quantitative measurements of peptides and proteins. The electrospray ionization efficiency is typically improved at nanospray flow rates due to the smaller size of the original droplets formed at the emitter tip.

However, single capillary-based nanospray lacks the speed and robustness that is required for many clinical and bio-pharmaceutical applications. One approach is to maintain microflow LC conditions while achieving nanoflow sensitivity by dividing the eluent from the LC system over a multiple of electrospray emitters [1], each operating at a fraction of the total liquid flow rate. The theoretical ion current increase is proportional to the square root of the number of emitters [2].

Methods Various samples were used to evaluate the multinozzle emitter arrays, provided by Newomics Inc. with exploratory infusion experiments at flow rates between 600 nL/min and 10 μL/min. The samples were chosen to cover several classes of compounds (small drug molecules, various proteins and peptides). Under standard nanospray conditions, these samples form singly as well as multiply charged protonated molecular ions, and were dissolved in solvent with different acidities to investigate the ionization process for the new multinozzle emitter arrays.

• “cal-mix”, a mixture of 2.0 ng/μL Caffeine, 5.5 pmol/μL MRFA (Met-Arg-Phe-Ala), and Ultramark 1621, dissolved in 1:1:2 methanol:water:acetonitrile (1.0% Acetic Acid)

• 1.0 pmol/μL Angiotensin I dissolved in 1:1 methanol:water (0.1% Acetic Acid) • 1.0 pmol/μL Hemoglobin A dissolved in 1:1 acetonitrile:water (0.2% Formic Acid)

A separate assay, including constituents of Bovine Serum Albumin (BSA) and Enolase diluted in E. Coli digest was used to characterize sensitivity at a nano-flow rate (300 nL/min). Six target constituents were resolved using a Thermo ScientificTM AcclaimTM PepMapTM RSLC 50μm x 15cm, C18, 2μm, 100 Å column over a 25 min duration gradient program controlled by a Thermo ScientificTM EASY-nLC™ 1000 system. Composition of the mobile phases was varied in multiple steps from 5% aqueous (0.1% Formic Acid) to 90% methanol (0.1% Formic Acid)

The assays were analyzed on the Thermo ScientificTM TSQ QuantivaTM triple-stage quadrupole mass spectrometer equipped with a slotted ion transfer tube held at 270 – 300 °C. The mass spectrometer was operated in the full scan mode at the 1000 Da/sec scan rate, or alternatively in the SRM mode with a 10 msec cycle time and the Argon collision gas pressure was 1.5 mTorr.

The multinozzle emitter array device was tightly clamped between a PEEK plate and a steel plate, where the PEEK plate has a threaded hole to accept standard fittings for capillary tubing and a gasket on the device side to prevent fluid leakage. The steel plate is connected to the HV power supply of the mass spectrometer (Figures 2d). This sandwiched assembly was mounted on a translation stage in front of the mass spectrometer inlet using a modified Thermo ScientificTM Nanospray Flex™ ion source (Figure 2e).

Establishing Taylor Cones on Multinozzle Emitter Arrays Experimental conditions were optimized to create a sufficiently high local electric field on the prototype multinozzle (n=1-100) emitter arrays to successfully form and sustain a uniform array of Taylor cones for a wide range of samples and flow rates without coalescence of the liquid as observed with an optical microscope (Figure 3). Note that at lower per nozzle flow rates and at smaller nozzle cross sections it was difficult to illuminate and observe the finer mist, hence the photographs presented here are at the high end of the flow rate range that was studied.

It can be observed in Figure 3 that the Taylor cones of the nozzles remained parallel with a remarkably small deviation at either end. Further, this deviation angle was found to increase with the liquid flow rate.

It is worth emphasizing that our emitter arrays allow for the stable operation at extremely high flow rates before eventually suffering the post nozzle coalescence often reported with alternative designs from others at much lower flow rates. The arrays with the larger 10 × 10 μm nozzles can be operated without backpressure limitations at flow rates that far exceed the normal nanoflow regime, while the individual nozzles still experience a flow rate well within or even on the low end of this range. For example, in Figure 4c, 15 μl/min was consumed with a low per nozzle flow rate of 300 nL/min. Potential applications are envisioned of the multinozzle emitter arrays for assays at microspray flow rates with reduced ion suppression.

The distance between the emitter array chip and the mass spectrometer inlet as well as the spray voltage were optimized in each experiment. It was found that compared to an uncoated pulled tip emitter (operated typically at 1.5 kV), a higher voltage was needed both for the onset of Taylor cones, as well as for optimal signal, with optimized values between 3.3 and 3.6 kV. The voltage needed to be increased up to the breakdown limit, a likely indication that for those number of emitters in the array, and under those flow conditions, the local electric field is close to being sufficient. A possible explanation would be the interference effects by the electric fields formed by neighboring emitters [5], and the current design perhaps could benefit from the inclusion of an extraction electrode to enhance the shielding between neighboring nozzles.

Optimum signal was found for those emitter arrays with overall widths that were not exceeding that of the slotted inlet of the mass spectrometer (i.e. 2.0 mm).

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

PO64083-EN 0614S

FIGURE 4. Total Ion Chromatograms (TIC) showing the stability of the signal in top panels (a)–(c) and, in bottom panels (a)–(c), the time integrated mass resolved spectra (Q1MS mode at 0.7 Da resolution) of Hemoglobin A (1.0 pmol/μL infusion) employing multinozzle emitter chips of 10 × 10 μm each: (a) 25-nozzle array at 3.1 kV at 600 nL/min (24 nL/min per nozzle); (b) 50-nozzle array at 3.6 kV at 600 nL/min (12 nL/min per nozzle); (c) 50-nozzle array at 3.4 kV at 2 μL/min (40 nL/min per nozzle). The base peak intensity as a function of total liquid flow rate is depicted for several emitter arrays (d).

Infusion at Nano and Micro Flow Rates The emitter array was found to produce short and long term stable peak intensities in the mass spectrometer (Figure 4).

It can also be observed in Figures 4a – 4c that the charge state distribution was not dependent number of nozzles or liquid flow rate. Additional studies (not shown) also established that it was independent of other device design parameters, and similar to that of a standard nanospray emitter.

Comparing the signal intensity for a fixed total liquid flow rate between emitter arrays with different number of nozzles with the same 10 × 10 μm cross section (Figure 4d), it can be seen that the emitter arrays show increased signal relative to a standard single uncoated pulled tip nanospray emitter (10 μm diameter).

The advantages provided by the photolithographic reproduction on chip-to-chip reproducibility can be judged by the similarity of signal at various flow rates of three 25-nozzle emitter arrays in Figure 4d.

Nanoflow LC-MS-MS Experiments A nanoflow chromatography SRM experiment was performed for the 25-nozzle (10 × 10 μm) emitter array by targeting six constituents of a mixture of BSA and Enolase in an E. Coli digest: TFAEALR (m/z 404.22), AEFVEVTK (m/z 461.75), IGSEVYHNLK (m/z 580.31), LVNELTEFAK (m/z 582.32), NVNDVIAPAFVK (m/z 643.86), and HLVDEPQNLIK (m/z 653.36). We found that the average peak intensities showed good reproducibility between replicate injections (Figure 5). At the concentrations studied this assay is not signal to noise limited and should well represent the number of ions generated.

The slight peak tailing observed in the chromatograms is indicative of post column dead volume, and we identified places to improve the interface to the LC. We are currently optimizing the connections between the multinozzle emitter arrays and LC column to minimize this dead volume.

Further experimentation is underway to compare emitter arrays with different number of nozzles with varying cross sections. For example, for total flow of 300 nL/min, the flow through one of the nozzles in a 50 emitter array is only 6 nL/min, and a 10 × 10 μm nozzle cross section geometry might be less than optimal. Further experiments with a smaller nozzle for the emitter arrays with larger number of nozzles are required.

Parameter Values Number of nozzles 1, 5, 10, 20, 40, 80 Total array width 1.0, 1.75, 2.0, 3.5 mm Nozzle cross section 3 × 3, 5 × 5, 10 × 10 μm Protrusion of nozzle above wafer ~100 μm Inter nozzle spacing 30, 35, 40, 60, 80, 100, 150, 200 μm

TABLE 1. Design parameters of the silicon-chip-based multinozzle emitter arrays. FIGURE 1. Cross section of the monolithic multinozzle emitter array chip. The chip was produced using standard silicon microchip fabrication techniques. Two wafers were bonded together to form an internal channel. In published experiments, this channel could be packed with different kinds (C4 and C18) of beads for on-chip LC separation.

FIGURE 3. Optical microscope images of separate Taylor cones formed by (a) 10-nozzle emitter array (10 × 10 μm nozzles) at a spray voltage of 2.8 kV and 3 μL/min infusion of Angiotensin I, i.e. a flow rate through each nozzle of 300 nL/min; (b) 25-nozzle emitter array (10 × 10 μm nozzles) at 3.5 kV spraying 2 μL/min of Hemoglobin A, i.e. a flow rate through each nozzle of 80 nL/min; (c) 50-nozzle emitter array (10 × 10 μm nozzles) at 4.2 kV spraying 15 μL/min of “cal-mix”, i.e. a flow rate through each nozzle of 300 nL/min; (d) 50-nozzle emitter array (3 × 3 μm nozzles) at 3.8 kV spraying 5 μL/min of acetonitrile (0.1% Formic Acid): 100 nL/min per nozzle. The area represented in the micrographs measures 3.0 × 2.0 mm.

(c)

(a)

2.0

mm

wid

e in

let

FIGURE 2. Scanning electron microscope images of sharpened-end multinozzle emitters: (a) 1-nozzle emitter with a cross section of 10 × 10 μm. (b) 20-nozzle emitter with a cross section of 5 x 5 μm. (c) 40-nozzle emitter with a cross section of 2 × 2.5 μm. The zoom-out and close-up views of each emitter are shown in the subpanels (i) and (ii), respectively. Photographs of (d) the multinozzle emitter mounted in a prototype holder, and (e) the holder mounted on the Nanospray Flex NG ion source in front of the slotted ion transfer tube inlet to the mass spectrometer.

d

e

a

b

c

FIGURE 5. Total Ion Chromatogram (TIC) showing the elution of six constituents from a mixture of BSA and Enolase at a flow rate of 300 nL/min. The bar charts on the right represent the peak area for three replicate on column injections of 10 femtomole using a 25-nozzle emitter array of 10 × 10 μm nozzles at 3.3 kV.

0.E+00

1.E+05

NVNDVIAPAFVK 0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05 LVNELTEFAK

0.0E+00

5.0E+04

1.0E+05 HLVDEPQNLIK

0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05 AEFVEVTK

NL: 3.73E4 TIC MS

IGSE

VYH

NLK

AEF

VEVT

K

HLV

DEP

QN

LIK

TFAE

ALR

LVN

ELTE

FAK

N

VND

VIAP

AFVK

BSA-Enolase-10femtomole-newomics-25-run2

13 14 15 16 17 18 19 20 21 Time (min)

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20

30

40

50

60

70

80

90

100

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(b) (a)

(c)

(b)

0.0E+00

5.0E+06

1.0E+07

1.5E+07

0 0.5 1 1.5 2 2.5

Bas

e pe

ak in

tens

ity

Total flow rate (μL/min)

25-emitter #1 25-emitter #2 25-emitter #3

50-emitter single pulled tip

(d)

4 Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Results Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays To overcome the limitations found in prior multisprayer designs, such as the chemical and thermal stability of the material [3], we have developed novel monolithic, silicon-chip-based, multinozzle emitter arrays [4].

These devices are produced using photolithography and deep reactive ion etching from two silicon wafers that are bonded together to form an internal channel (Figure 1). All surfaces that surround the channel are wet oxidized to form a thick oxide layer, and the resulting protruding nozzles have a square cross section profile (Figure 2a-c) and constituted of SiO2. The standard silicon microchip fabrication technology ensures reproducibility between devices as well as the potential of creating of a large number of small high aspect ratio structures that allow for the creation of a local electric field strength strong enough to form a Taylor cone.

The multinozzle emitter array chips also offer a straightforward solution to the problem of splitting the liquid flow without leaks or dead volume, which could prevent their practical use. The internal channel of the chip is 20 mm long, 100 μm wide, and has a depth of 10 μm (volume is 20 nL), and can in the future be packed with resin beads to perform on-chip LC separations.

The issue of the poor geometric overlap of a linear array of emitters with the circular atmospheric inlet to a mass spectrometer, as we described in Ref. 4, has been addressed by employing a rectangular slotted ion transfer tube that was perfectly aligned with the linear emitter array.

Table 1 lists the device geometry design parameters that have been varied during the microchip production process: emitter tip spacing, protrusion, emitter channel size, and number of emitters ranging from 1 to 80. A small selection of these microchip emitter arrays has so far been experimentally investigated.

Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Eloy R. Wouters1, Pan Mao2, Jean-Jacques Dunyach1, Daojing Wang2 1Thermo Fisher Scientific, San Jose, CA, 2Newomics Inc., Emeryville, CA

Conclusion Multinozzle emitter arrays were fabricated from a monolithic silicon substrate,

creating sufficient electric field strength to form and sustain a stable uniform array of Taylor cones without coalescence of the fluid as well as reproducible sensitivity between emitter chips

Linear geometry of the multinozzle emitter array makes feasible the optimal geometric overlap with the slotted inlet on the TSQ QuantivaTM triple quadrupole mass spectrometer

Possible solution for microflow electrospray applications with reduced ion suppression

Intensity gains observed in infusion

Further experimentation is ongoing: to find the optimal nozzle cross section for each number of nozzles to evaluate the potential for increased linearity of the ion source and more

uniform ionization efficiency for different analytes to establish overall ruggedness of the device for LC-MS-MS applications

with samples in biological matrices which is a key parameter for its success

Future directions include the stacking of several arrays to increase the number of nozzle per emitter while retaining the geometric overlap with the slotted atmospheric pressure inlet.

References 1. A.J. Rulison and R.C. Flagan, Scale-up of Electrospray Atomization Using Linear

Arrays of Taylor Cones, Rev. Sci. Instrum. 64, 683 (1993)

2. J.F. de la Mora and I.G. Loscertales, The Current Emitted by Highly Conducting Taylor Cones, J. Fluid Mech. 260, 155 (1994)

3. K. Tang, Y. Lin, D.W. Matson, T. Kim, and R.D. Smith, Generation of Multiple Electrosprays Using Microfabricated Emitters Arrays for Improved Mass Spectrometric Sensitivity, Anal. Chem. 73, 1658 (2001)

4. P. Mao, H-T. Wang, P. Yang, and D. Wang, Multinozzle Emitter Arrays for Nanoelectrospray Mass Spectrometry, Anal. Chem. 83, 6082 (2011)

5. B.Q.T. Si, D. Byun, and S. Lee, Experimental and Theoretical Study of a Cone-jet for an Electrospray Microthruster Considering the Interference Effect in an Array of Nozzles, Aerosol Science 38, 924 (2007)

Acknowledgements Financial support from NIH SBIR program to Newomics Inc. is gratefully acknowledged. We thank the UC Berkeley Marvell Nanofabrication Laboratory for facility access.

Overview Purpose: Evaluation of linear multinozzle emitter arrays for low flow mass spectroscopy.

Methods: Infusion at microflow rates and LC-MS-MS at nanoflow rates on a triple quadrupole mass spectrometer equipped with a slotted tube.

Results: Successful generation of a large number of Taylor cones and stable ion signal over a wide range of liquid flow rates.

Introduction The soft ionization provided by nano-electrospray sources used in mass spectrometry is an important tool for qualitative and quantitative measurements of peptides and proteins. The electrospray ionization efficiency is typically improved at nanospray flow rates due to the smaller size of the original droplets formed at the emitter tip.

However, single capillary-based nanospray lacks the speed and robustness that is required for many clinical and bio-pharmaceutical applications. One approach is to maintain microflow LC conditions while achieving nanoflow sensitivity by dividing the eluent from the LC system over a multiple of electrospray emitters [1], each operating at a fraction of the total liquid flow rate. The theoretical ion current increase is proportional to the square root of the number of emitters [2].

Methods Various samples were used to evaluate the multinozzle emitter arrays, provided by Newomics Inc. with exploratory infusion experiments at flow rates between 600 nL/min and 10 μL/min. The samples were chosen to cover several classes of compounds (small drug molecules, various proteins and peptides). Under standard nanospray conditions, these samples form singly as well as multiply charged protonated molecular ions, and were dissolved in solvent with different acidities to investigate the ionization process for the new multinozzle emitter arrays.

• “cal-mix”, a mixture of 2.0 ng/μL Caffeine, 5.5 pmol/μL MRFA (Met-Arg-Phe-Ala), and Ultramark 1621, dissolved in 1:1:2 methanol:water:acetonitrile (1.0% Acetic Acid)

• 1.0 pmol/μL Angiotensin I dissolved in 1:1 methanol:water (0.1% Acetic Acid) • 1.0 pmol/μL Hemoglobin A dissolved in 1:1 acetonitrile:water (0.2% Formic Acid)

A separate assay, including constituents of Bovine Serum Albumin (BSA) and Enolase diluted in E. Coli digest was used to characterize sensitivity at a nano-flow rate (300 nL/min). Six target constituents were resolved using a Thermo ScientificTM AcclaimTM PepMapTM RSLC 50μm x 15cm, C18, 2μm, 100 Å column over a 25 min duration gradient program controlled by a Thermo ScientificTM EASY-nLC™ 1000 system. Composition of the mobile phases was varied in multiple steps from 5% aqueous (0.1% Formic Acid) to 90% methanol (0.1% Formic Acid)

The assays were analyzed on the Thermo ScientificTM TSQ QuantivaTM triple-stage quadrupole mass spectrometer equipped with a slotted ion transfer tube held at 270 – 300 °C. The mass spectrometer was operated in the full scan mode at the 1000 Da/sec scan rate, or alternatively in the SRM mode with a 10 msec cycle time and the Argon collision gas pressure was 1.5 mTorr.

The multinozzle emitter array device was tightly clamped between a PEEK plate and a steel plate, where the PEEK plate has a threaded hole to accept standard fittings for capillary tubing and a gasket on the device side to prevent fluid leakage. The steel plate is connected to the HV power supply of the mass spectrometer (Figures 2d). This sandwiched assembly was mounted on a translation stage in front of the mass spectrometer inlet using a modified Thermo ScientificTM Nanospray Flex™ ion source (Figure 2e).

Establishing Taylor Cones on Multinozzle Emitter Arrays Experimental conditions were optimized to create a sufficiently high local electric field on the prototype multinozzle (n=1-100) emitter arrays to successfully form and sustain a uniform array of Taylor cones for a wide range of samples and flow rates without coalescence of the liquid as observed with an optical microscope (Figure 3). Note that at lower per nozzle flow rates and at smaller nozzle cross sections it was difficult to illuminate and observe the finer mist, hence the photographs presented here are at the high end of the flow rate range that was studied.

It can be observed in Figure 3 that the Taylor cones of the nozzles remained parallel with a remarkably small deviation at either end. Further, this deviation angle was found to increase with the liquid flow rate.

It is worth emphasizing that our emitter arrays allow for the stable operation at extremely high flow rates before eventually suffering the post nozzle coalescence often reported with alternative designs from others at much lower flow rates. The arrays with the larger 10 × 10 μm nozzles can be operated without backpressure limitations at flow rates that far exceed the normal nanoflow regime, while the individual nozzles still experience a flow rate well within or even on the low end of this range. For example, in Figure 4c, 15 μl/min was consumed with a low per nozzle flow rate of 300 nL/min. Potential applications are envisioned of the multinozzle emitter arrays for assays at microspray flow rates with reduced ion suppression.

The distance between the emitter array chip and the mass spectrometer inlet as well as the spray voltage were optimized in each experiment. It was found that compared to an uncoated pulled tip emitter (operated typically at 1.5 kV), a higher voltage was needed both for the onset of Taylor cones, as well as for optimal signal, with optimized values between 3.3 and 3.6 kV. The voltage needed to be increased up to the breakdown limit, a likely indication that for those number of emitters in the array, and under those flow conditions, the local electric field is close to being sufficient. A possible explanation would be the interference effects by the electric fields formed by neighboring emitters [5], and the current design perhaps could benefit from the inclusion of an extraction electrode to enhance the shielding between neighboring nozzles.

Optimum signal was found for those emitter arrays with overall widths that were not exceeding that of the slotted inlet of the mass spectrometer (i.e. 2.0 mm).

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

PO64083-EN 0614S

FIGURE 4. Total Ion Chromatograms (TIC) showing the stability of the signal in top panels (a)–(c) and, in bottom panels (a)–(c), the time integrated mass resolved spectra (Q1MS mode at 0.7 Da resolution) of Hemoglobin A (1.0 pmol/μL infusion) employing multinozzle emitter chips of 10 × 10 μm each: (a) 25-nozzle array at 3.1 kV at 600 nL/min (24 nL/min per nozzle); (b) 50-nozzle array at 3.6 kV at 600 nL/min (12 nL/min per nozzle); (c) 50-nozzle array at 3.4 kV at 2 μL/min (40 nL/min per nozzle). The base peak intensity as a function of total liquid flow rate is depicted for several emitter arrays (d).

Infusion at Nano and Micro Flow Rates The emitter array was found to produce short and long term stable peak intensities in the mass spectrometer (Figure 4).

It can also be observed in Figures 4a – 4c that the charge state distribution was not dependent number of nozzles or liquid flow rate. Additional studies (not shown) also established that it was independent of other device design parameters, and similar to that of a standard nanospray emitter.

Comparing the signal intensity for a fixed total liquid flow rate between emitter arrays with different number of nozzles with the same 10 × 10 μm cross section (Figure 4d), it can be seen that the emitter arrays show increased signal relative to a standard single uncoated pulled tip nanospray emitter (10 μm diameter).

The advantages provided by the photolithographic reproduction on chip-to-chip reproducibility can be judged by the similarity of signal at various flow rates of three 25-nozzle emitter arrays in Figure 4d.

Nanoflow LC-MS-MS Experiments A nanoflow chromatography SRM experiment was performed for the 25-nozzle (10 × 10 μm) emitter array by targeting six constituents of a mixture of BSA and Enolase in an E. Coli digest: TFAEALR (m/z 404.22), AEFVEVTK (m/z 461.75), IGSEVYHNLK (m/z 580.31), LVNELTEFAK (m/z 582.32), NVNDVIAPAFVK (m/z 643.86), and HLVDEPQNLIK (m/z 653.36). We found that the average peak intensities showed good reproducibility between replicate injections (Figure 5). At the concentrations studied this assay is not signal to noise limited and should well represent the number of ions generated.

The slight peak tailing observed in the chromatograms is indicative of post column dead volume, and we identified places to improve the interface to the LC. We are currently optimizing the connections between the multinozzle emitter arrays and LC column to minimize this dead volume.

Further experimentation is underway to compare emitter arrays with different number of nozzles with varying cross sections. For example, for total flow of 300 nL/min, the flow through one of the nozzles in a 50 emitter array is only 6 nL/min, and a 10 × 10 μm nozzle cross section geometry might be less than optimal. Further experiments with a smaller nozzle for the emitter arrays with larger number of nozzles are required.

Parameter Values Number of nozzles 1, 5, 10, 20, 40, 80 Total array width 1.0, 1.75, 2.0, 3.5 mm Nozzle cross section 3 × 3, 5 × 5, 10 × 10 μm Protrusion of nozzle above wafer ~100 μm Inter nozzle spacing 30, 35, 40, 60, 80, 100, 150, 200 μm

TABLE 1. Design parameters of the silicon-chip-based multinozzle emitter arrays. FIGURE 1. Cross section of the monolithic multinozzle emitter array chip. The chip was produced using standard silicon microchip fabrication techniques. Two wafers were bonded together to form an internal channel. In published experiments, this channel could be packed with different kinds (C4 and C18) of beads for on-chip LC separation.

FIGURE 3. Optical microscope images of separate Taylor cones formed by (a) 10-nozzle emitter array (10 × 10 μm nozzles) at a spray voltage of 2.8 kV and 3 μL/min infusion of Angiotensin I, i.e. a flow rate through each nozzle of 300 nL/min; (b) 25-nozzle emitter array (10 × 10 μm nozzles) at 3.5 kV spraying 2 μL/min of Hemoglobin A, i.e. a flow rate through each nozzle of 80 nL/min; (c) 50-nozzle emitter array (10 × 10 μm nozzles) at 4.2 kV spraying 15 μL/min of “cal-mix”, i.e. a flow rate through each nozzle of 300 nL/min; (d) 50-nozzle emitter array (3 × 3 μm nozzles) at 3.8 kV spraying 5 μL/min of acetonitrile (0.1% Formic Acid): 100 nL/min per nozzle. The area represented in the micrographs measures 3.0 × 2.0 mm.

(c)

(a)

2.0

mm

wid

e in

let

FIGURE 2. Scanning electron microscope images of sharpened-end multinozzle emitters: (a) 1-nozzle emitter with a cross section of 10 × 10 μm. (b) 20-nozzle emitter with a cross section of 5 x 5 μm. (c) 40-nozzle emitter with a cross section of 2 × 2.5 μm. The zoom-out and close-up views of each emitter are shown in the subpanels (i) and (ii), respectively. Photographs of (d) the multinozzle emitter mounted in a prototype holder, and (e) the holder mounted on the Nanospray Flex NG ion source in front of the slotted ion transfer tube inlet to the mass spectrometer.

d

e

a

b

c

FIGURE 5. Total Ion Chromatogram (TIC) showing the elution of six constituents from a mixture of BSA and Enolase at a flow rate of 300 nL/min. The bar charts on the right represent the peak area for three replicate on column injections of 10 femtomole using a 25-nozzle emitter array of 10 × 10 μm nozzles at 3.3 kV.

0.E+00

1.E+05

NVNDVIAPAFVK 0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05 LVNELTEFAK

0.0E+00

5.0E+04

1.0E+05 HLVDEPQNLIK

0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05 AEFVEVTK

NL: 3.73E4 TIC MS

IGSE

VYH

NLK

AEF

VEVT

K

HLV

DEP

QN

LIK

TFAE

ALR

LVN

ELTE

FAK

N

VND

VIAP

AFVK

BSA-Enolase-10femtomole-newomics-25-run2

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25-emitter #1 25-emitter #2 25-emitter #3

50-emitter single pulled tip

(d)

5Thermo Scientific Poster Note • PN-64083-ASMS-EN-0614S

Results Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays To overcome the limitations found in prior multisprayer designs, such as the chemical and thermal stability of the material [3], we have developed novel monolithic, silicon-chip-based, multinozzle emitter arrays [4].

These devices are produced using photolithography and deep reactive ion etching from two silicon wafers that are bonded together to form an internal channel (Figure 1). All surfaces that surround the channel are wet oxidized to form a thick oxide layer, and the resulting protruding nozzles have a square cross section profile (Figure 2a-c) and constituted of SiO2. The standard silicon microchip fabrication technology ensures reproducibility between devices as well as the potential of creating of a large number of small high aspect ratio structures that allow for the creation of a local electric field strength strong enough to form a Taylor cone.

The multinozzle emitter array chips also offer a straightforward solution to the problem of splitting the liquid flow without leaks or dead volume, which could prevent their practical use. The internal channel of the chip is 20 mm long, 100 μm wide, and has a depth of 10 μm (volume is 20 nL), and can in the future be packed with resin beads to perform on-chip LC separations.

The issue of the poor geometric overlap of a linear array of emitters with the circular atmospheric inlet to a mass spectrometer, as we described in Ref. 4, has been addressed by employing a rectangular slotted ion transfer tube that was perfectly aligned with the linear emitter array.

Table 1 lists the device geometry design parameters that have been varied during the microchip production process: emitter tip spacing, protrusion, emitter channel size, and number of emitters ranging from 1 to 80. A small selection of these microchip emitter arrays has so far been experimentally investigated.

Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Eloy R. Wouters1, Pan Mao2, Jean-Jacques Dunyach1, Daojing Wang2 1Thermo Fisher Scientific, San Jose, CA, 2Newomics Inc., Emeryville, CA

Conclusion Multinozzle emitter arrays were fabricated from a monolithic silicon substrate,

creating sufficient electric field strength to form and sustain a stable uniform array of Taylor cones without coalescence of the fluid as well as reproducible sensitivity between emitter chips

Linear geometry of the multinozzle emitter array makes feasible the optimal geometric overlap with the slotted inlet on the TSQ QuantivaTM triple quadrupole mass spectrometer

Possible solution for microflow electrospray applications with reduced ion suppression

Intensity gains observed in infusion

Further experimentation is ongoing: to find the optimal nozzle cross section for each number of nozzles to evaluate the potential for increased linearity of the ion source and more

uniform ionization efficiency for different analytes to establish overall ruggedness of the device for LC-MS-MS applications

with samples in biological matrices which is a key parameter for its success

Future directions include the stacking of several arrays to increase the number of nozzle per emitter while retaining the geometric overlap with the slotted atmospheric pressure inlet.

References 1. A.J. Rulison and R.C. Flagan, Scale-up of Electrospray Atomization Using Linear

Arrays of Taylor Cones, Rev. Sci. Instrum. 64, 683 (1993)

2. J.F. de la Mora and I.G. Loscertales, The Current Emitted by Highly Conducting Taylor Cones, J. Fluid Mech. 260, 155 (1994)

3. K. Tang, Y. Lin, D.W. Matson, T. Kim, and R.D. Smith, Generation of Multiple Electrosprays Using Microfabricated Emitters Arrays for Improved Mass Spectrometric Sensitivity, Anal. Chem. 73, 1658 (2001)

4. P. Mao, H-T. Wang, P. Yang, and D. Wang, Multinozzle Emitter Arrays for Nanoelectrospray Mass Spectrometry, Anal. Chem. 83, 6082 (2011)

5. B.Q.T. Si, D. Byun, and S. Lee, Experimental and Theoretical Study of a Cone-jet for an Electrospray Microthruster Considering the Interference Effect in an Array of Nozzles, Aerosol Science 38, 924 (2007)

Acknowledgements Financial support from NIH SBIR program to Newomics Inc. is gratefully acknowledged. We thank the UC Berkeley Marvell Nanofabrication Laboratory for facility access.

Overview Purpose: Evaluation of linear multinozzle emitter arrays for low flow mass spectroscopy.

Methods: Infusion at microflow rates and LC-MS-MS at nanoflow rates on a triple quadrupole mass spectrometer equipped with a slotted tube.

Results: Successful generation of a large number of Taylor cones and stable ion signal over a wide range of liquid flow rates.

Introduction The soft ionization provided by nano-electrospray sources used in mass spectrometry is an important tool for qualitative and quantitative measurements of peptides and proteins. The electrospray ionization efficiency is typically improved at nanospray flow rates due to the smaller size of the original droplets formed at the emitter tip.

However, single capillary-based nanospray lacks the speed and robustness that is required for many clinical and bio-pharmaceutical applications. One approach is to maintain microflow LC conditions while achieving nanoflow sensitivity by dividing the eluent from the LC system over a multiple of electrospray emitters [1], each operating at a fraction of the total liquid flow rate. The theoretical ion current increase is proportional to the square root of the number of emitters [2].

Methods Various samples were used to evaluate the multinozzle emitter arrays, provided by Newomics Inc. with exploratory infusion experiments at flow rates between 600 nL/min and 10 μL/min. The samples were chosen to cover several classes of compounds (small drug molecules, various proteins and peptides). Under standard nanospray conditions, these samples form singly as well as multiply charged protonated molecular ions, and were dissolved in solvent with different acidities to investigate the ionization process for the new multinozzle emitter arrays.

• “cal-mix”, a mixture of 2.0 ng/μL Caffeine, 5.5 pmol/μL MRFA (Met-Arg-Phe-Ala), and Ultramark 1621, dissolved in 1:1:2 methanol:water:acetonitrile (1.0% Acetic Acid)

• 1.0 pmol/μL Angiotensin I dissolved in 1:1 methanol:water (0.1% Acetic Acid) • 1.0 pmol/μL Hemoglobin A dissolved in 1:1 acetonitrile:water (0.2% Formic Acid)

A separate assay, including constituents of Bovine Serum Albumin (BSA) and Enolase diluted in E. Coli digest was used to characterize sensitivity at a nano-flow rate (300 nL/min). Six target constituents were resolved using a Thermo ScientificTM AcclaimTM PepMapTM RSLC 50μm x 15cm, C18, 2μm, 100 Å column over a 25 min duration gradient program controlled by a Thermo ScientificTM EASY-nLC™ 1000 system. Composition of the mobile phases was varied in multiple steps from 5% aqueous (0.1% Formic Acid) to 90% methanol (0.1% Formic Acid)

The assays were analyzed on the Thermo ScientificTM TSQ QuantivaTM triple-stage quadrupole mass spectrometer equipped with a slotted ion transfer tube held at 270 – 300 °C. The mass spectrometer was operated in the full scan mode at the 1000 Da/sec scan rate, or alternatively in the SRM mode with a 10 msec cycle time and the Argon collision gas pressure was 1.5 mTorr.

The multinozzle emitter array device was tightly clamped between a PEEK plate and a steel plate, where the PEEK plate has a threaded hole to accept standard fittings for capillary tubing and a gasket on the device side to prevent fluid leakage. The steel plate is connected to the HV power supply of the mass spectrometer (Figures 2d). This sandwiched assembly was mounted on a translation stage in front of the mass spectrometer inlet using a modified Thermo ScientificTM Nanospray Flex™ ion source (Figure 2e).

Establishing Taylor Cones on Multinozzle Emitter Arrays Experimental conditions were optimized to create a sufficiently high local electric field on the prototype multinozzle (n=1-100) emitter arrays to successfully form and sustain a uniform array of Taylor cones for a wide range of samples and flow rates without coalescence of the liquid as observed with an optical microscope (Figure 3). Note that at lower per nozzle flow rates and at smaller nozzle cross sections it was difficult to illuminate and observe the finer mist, hence the photographs presented here are at the high end of the flow rate range that was studied.

It can be observed in Figure 3 that the Taylor cones of the nozzles remained parallel with a remarkably small deviation at either end. Further, this deviation angle was found to increase with the liquid flow rate.

It is worth emphasizing that our emitter arrays allow for the stable operation at extremely high flow rates before eventually suffering the post nozzle coalescence often reported with alternative designs from others at much lower flow rates. The arrays with the larger 10 × 10 μm nozzles can be operated without backpressure limitations at flow rates that far exceed the normal nanoflow regime, while the individual nozzles still experience a flow rate well within or even on the low end of this range. For example, in Figure 4c, 15 μl/min was consumed with a low per nozzle flow rate of 300 nL/min. Potential applications are envisioned of the multinozzle emitter arrays for assays at microspray flow rates with reduced ion suppression.

The distance between the emitter array chip and the mass spectrometer inlet as well as the spray voltage were optimized in each experiment. It was found that compared to an uncoated pulled tip emitter (operated typically at 1.5 kV), a higher voltage was needed both for the onset of Taylor cones, as well as for optimal signal, with optimized values between 3.3 and 3.6 kV. The voltage needed to be increased up to the breakdown limit, a likely indication that for those number of emitters in the array, and under those flow conditions, the local electric field is close to being sufficient. A possible explanation would be the interference effects by the electric fields formed by neighboring emitters [5], and the current design perhaps could benefit from the inclusion of an extraction electrode to enhance the shielding between neighboring nozzles.

Optimum signal was found for those emitter arrays with overall widths that were not exceeding that of the slotted inlet of the mass spectrometer (i.e. 2.0 mm).

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

PO64083-EN 0614S

FIGURE 4. Total Ion Chromatograms (TIC) showing the stability of the signal in top panels (a)–(c) and, in bottom panels (a)–(c), the time integrated mass resolved spectra (Q1MS mode at 0.7 Da resolution) of Hemoglobin A (1.0 pmol/μL infusion) employing multinozzle emitter chips of 10 × 10 μm each: (a) 25-nozzle array at 3.1 kV at 600 nL/min (24 nL/min per nozzle); (b) 50-nozzle array at 3.6 kV at 600 nL/min (12 nL/min per nozzle); (c) 50-nozzle array at 3.4 kV at 2 μL/min (40 nL/min per nozzle). The base peak intensity as a function of total liquid flow rate is depicted for several emitter arrays (d).

Infusion at Nano and Micro Flow Rates The emitter array was found to produce short and long term stable peak intensities in the mass spectrometer (Figure 4).

It can also be observed in Figures 4a – 4c that the charge state distribution was not dependent number of nozzles or liquid flow rate. Additional studies (not shown) also established that it was independent of other device design parameters, and similar to that of a standard nanospray emitter.

Comparing the signal intensity for a fixed total liquid flow rate between emitter arrays with different number of nozzles with the same 10 × 10 μm cross section (Figure 4d), it can be seen that the emitter arrays show increased signal relative to a standard single uncoated pulled tip nanospray emitter (10 μm diameter).

The advantages provided by the photolithographic reproduction on chip-to-chip reproducibility can be judged by the similarity of signal at various flow rates of three 25-nozzle emitter arrays in Figure 4d.

Nanoflow LC-MS-MS Experiments A nanoflow chromatography SRM experiment was performed for the 25-nozzle (10 × 10 μm) emitter array by targeting six constituents of a mixture of BSA and Enolase in an E. Coli digest: TFAEALR (m/z 404.22), AEFVEVTK (m/z 461.75), IGSEVYHNLK (m/z 580.31), LVNELTEFAK (m/z 582.32), NVNDVIAPAFVK (m/z 643.86), and HLVDEPQNLIK (m/z 653.36). We found that the average peak intensities showed good reproducibility between replicate injections (Figure 5). At the concentrations studied this assay is not signal to noise limited and should well represent the number of ions generated.

The slight peak tailing observed in the chromatograms is indicative of post column dead volume, and we identified places to improve the interface to the LC. We are currently optimizing the connections between the multinozzle emitter arrays and LC column to minimize this dead volume.

Further experimentation is underway to compare emitter arrays with different number of nozzles with varying cross sections. For example, for total flow of 300 nL/min, the flow through one of the nozzles in a 50 emitter array is only 6 nL/min, and a 10 × 10 μm nozzle cross section geometry might be less than optimal. Further experiments with a smaller nozzle for the emitter arrays with larger number of nozzles are required.

Parameter Values Number of nozzles 1, 5, 10, 20, 40, 80 Total array width 1.0, 1.75, 2.0, 3.5 mm Nozzle cross section 3 × 3, 5 × 5, 10 × 10 μm Protrusion of nozzle above wafer ~100 μm Inter nozzle spacing 30, 35, 40, 60, 80, 100, 150, 200 μm

TABLE 1. Design parameters of the silicon-chip-based multinozzle emitter arrays. FIGURE 1. Cross section of the monolithic multinozzle emitter array chip. The chip was produced using standard silicon microchip fabrication techniques. Two wafers were bonded together to form an internal channel. In published experiments, this channel could be packed with different kinds (C4 and C18) of beads for on-chip LC separation.

FIGURE 3. Optical microscope images of separate Taylor cones formed by (a) 10-nozzle emitter array (10 × 10 μm nozzles) at a spray voltage of 2.8 kV and 3 μL/min infusion of Angiotensin I, i.e. a flow rate through each nozzle of 300 nL/min; (b) 25-nozzle emitter array (10 × 10 μm nozzles) at 3.5 kV spraying 2 μL/min of Hemoglobin A, i.e. a flow rate through each nozzle of 80 nL/min; (c) 50-nozzle emitter array (10 × 10 μm nozzles) at 4.2 kV spraying 15 μL/min of “cal-mix”, i.e. a flow rate through each nozzle of 300 nL/min; (d) 50-nozzle emitter array (3 × 3 μm nozzles) at 3.8 kV spraying 5 μL/min of acetonitrile (0.1% Formic Acid): 100 nL/min per nozzle. The area represented in the micrographs measures 3.0 × 2.0 mm.

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FIGURE 2. Scanning electron microscope images of sharpened-end multinozzle emitters: (a) 1-nozzle emitter with a cross section of 10 × 10 μm. (b) 20-nozzle emitter with a cross section of 5 x 5 μm. (c) 40-nozzle emitter with a cross section of 2 × 2.5 μm. The zoom-out and close-up views of each emitter are shown in the subpanels (i) and (ii), respectively. Photographs of (d) the multinozzle emitter mounted in a prototype holder, and (e) the holder mounted on the Nanospray Flex NG ion source in front of the slotted ion transfer tube inlet to the mass spectrometer.

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FIGURE 5. Total Ion Chromatogram (TIC) showing the elution of six constituents from a mixture of BSA and Enolase at a flow rate of 300 nL/min. The bar charts on the right represent the peak area for three replicate on column injections of 10 femtomole using a 25-nozzle emitter array of 10 × 10 μm nozzles at 3.3 kV.

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6 Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Results Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays To overcome the limitations found in prior multisprayer designs, such as the chemical and thermal stability of the material [3], we have developed novel monolithic, silicon-chip-based, multinozzle emitter arrays [4].

These devices are produced using photolithography and deep reactive ion etching from two silicon wafers that are bonded together to form an internal channel (Figure 1). All surfaces that surround the channel are wet oxidized to form a thick oxide layer, and the resulting protruding nozzles have a square cross section profile (Figure 2a-c) and constituted of SiO2. The standard silicon microchip fabrication technology ensures reproducibility between devices as well as the potential of creating of a large number of small high aspect ratio structures that allow for the creation of a local electric field strength strong enough to form a Taylor cone.

The multinozzle emitter array chips also offer a straightforward solution to the problem of splitting the liquid flow without leaks or dead volume, which could prevent their practical use. The internal channel of the chip is 20 mm long, 100 μm wide, and has a depth of 10 μm (volume is 20 nL), and can in the future be packed with resin beads to perform on-chip LC separations.

The issue of the poor geometric overlap of a linear array of emitters with the circular atmospheric inlet to a mass spectrometer, as we described in Ref. 4, has been addressed by employing a rectangular slotted ion transfer tube that was perfectly aligned with the linear emitter array.

Table 1 lists the device geometry design parameters that have been varied during the microchip production process: emitter tip spacing, protrusion, emitter channel size, and number of emitters ranging from 1 to 80. A small selection of these microchip emitter arrays has so far been experimentally investigated.

Evaluation of Monolithic Silicon-Chip-Based Multinozzle Emitter Arrays for Nano- and Micro-Electrospray Mass Spectrometry

Eloy R. Wouters1, Pan Mao2, Jean-Jacques Dunyach1, Daojing Wang2 1Thermo Fisher Scientific, San Jose, CA, 2Newomics Inc., Emeryville, CA

Conclusion Multinozzle emitter arrays were fabricated from a monolithic silicon substrate,

creating sufficient electric field strength to form and sustain a stable uniform array of Taylor cones without coalescence of the fluid as well as reproducible sensitivity between emitter chips

Linear geometry of the multinozzle emitter array makes feasible the optimal geometric overlap with the slotted inlet on the TSQ QuantivaTM triple quadrupole mass spectrometer

Possible solution for microflow electrospray applications with reduced ion suppression

Intensity gains observed in infusion

Further experimentation is ongoing: to find the optimal nozzle cross section for each number of nozzles to evaluate the potential for increased linearity of the ion source and more

uniform ionization efficiency for different analytes to establish overall ruggedness of the device for LC-MS-MS applications

with samples in biological matrices which is a key parameter for its success

Future directions include the stacking of several arrays to increase the number of nozzle per emitter while retaining the geometric overlap with the slotted atmospheric pressure inlet.

References 1. A.J. Rulison and R.C. Flagan, Scale-up of Electrospray Atomization Using Linear

Arrays of Taylor Cones, Rev. Sci. Instrum. 64, 683 (1993)

2. J.F. de la Mora and I.G. Loscertales, The Current Emitted by Highly Conducting Taylor Cones, J. Fluid Mech. 260, 155 (1994)

3. K. Tang, Y. Lin, D.W. Matson, T. Kim, and R.D. Smith, Generation of Multiple Electrosprays Using Microfabricated Emitters Arrays for Improved Mass Spectrometric Sensitivity, Anal. Chem. 73, 1658 (2001)

4. P. Mao, H-T. Wang, P. Yang, and D. Wang, Multinozzle Emitter Arrays for Nanoelectrospray Mass Spectrometry, Anal. Chem. 83, 6082 (2011)

5. B.Q.T. Si, D. Byun, and S. Lee, Experimental and Theoretical Study of a Cone-jet for an Electrospray Microthruster Considering the Interference Effect in an Array of Nozzles, Aerosol Science 38, 924 (2007)

Acknowledgements Financial support from NIH SBIR program to Newomics Inc. is gratefully acknowledged. We thank the UC Berkeley Marvell Nanofabrication Laboratory for facility access.

Overview Purpose: Evaluation of linear multinozzle emitter arrays for low flow mass spectroscopy.

Methods: Infusion at microflow rates and LC-MS-MS at nanoflow rates on a triple quadrupole mass spectrometer equipped with a slotted tube.

Results: Successful generation of a large number of Taylor cones and stable ion signal over a wide range of liquid flow rates.

Introduction The soft ionization provided by nano-electrospray sources used in mass spectrometry is an important tool for qualitative and quantitative measurements of peptides and proteins. The electrospray ionization efficiency is typically improved at nanospray flow rates due to the smaller size of the original droplets formed at the emitter tip.

However, single capillary-based nanospray lacks the speed and robustness that is required for many clinical and bio-pharmaceutical applications. One approach is to maintain microflow LC conditions while achieving nanoflow sensitivity by dividing the eluent from the LC system over a multiple of electrospray emitters [1], each operating at a fraction of the total liquid flow rate. The theoretical ion current increase is proportional to the square root of the number of emitters [2].

Methods Various samples were used to evaluate the multinozzle emitter arrays, provided by Newomics Inc. with exploratory infusion experiments at flow rates between 600 nL/min and 10 μL/min. The samples were chosen to cover several classes of compounds (small drug molecules, various proteins and peptides). Under standard nanospray conditions, these samples form singly as well as multiply charged protonated molecular ions, and were dissolved in solvent with different acidities to investigate the ionization process for the new multinozzle emitter arrays.

• “cal-mix”, a mixture of 2.0 ng/μL Caffeine, 5.5 pmol/μL MRFA (Met-Arg-Phe-Ala), and Ultramark 1621, dissolved in 1:1:2 methanol:water:acetonitrile (1.0% Acetic Acid)

• 1.0 pmol/μL Angiotensin I dissolved in 1:1 methanol:water (0.1% Acetic Acid) • 1.0 pmol/μL Hemoglobin A dissolved in 1:1 acetonitrile:water (0.2% Formic Acid)

A separate assay, including constituents of Bovine Serum Albumin (BSA) and Enolase diluted in E. Coli digest was used to characterize sensitivity at a nano-flow rate (300 nL/min). Six target constituents were resolved using a Thermo ScientificTM AcclaimTM PepMapTM RSLC 50μm x 15cm, C18, 2μm, 100 Å column over a 25 min duration gradient program controlled by a Thermo ScientificTM EASY-nLC™ 1000 system. Composition of the mobile phases was varied in multiple steps from 5% aqueous (0.1% Formic Acid) to 90% methanol (0.1% Formic Acid)

The assays were analyzed on the Thermo ScientificTM TSQ QuantivaTM triple-stage quadrupole mass spectrometer equipped with a slotted ion transfer tube held at 270 – 300 °C. The mass spectrometer was operated in the full scan mode at the 1000 Da/sec scan rate, or alternatively in the SRM mode with a 10 msec cycle time and the Argon collision gas pressure was 1.5 mTorr.

The multinozzle emitter array device was tightly clamped between a PEEK plate and a steel plate, where the PEEK plate has a threaded hole to accept standard fittings for capillary tubing and a gasket on the device side to prevent fluid leakage. The steel plate is connected to the HV power supply of the mass spectrometer (Figures 2d). This sandwiched assembly was mounted on a translation stage in front of the mass spectrometer inlet using a modified Thermo ScientificTM Nanospray Flex™ ion source (Figure 2e).

Establishing Taylor Cones on Multinozzle Emitter Arrays Experimental conditions were optimized to create a sufficiently high local electric field on the prototype multinozzle (n=1-100) emitter arrays to successfully form and sustain a uniform array of Taylor cones for a wide range of samples and flow rates without coalescence of the liquid as observed with an optical microscope (Figure 3). Note that at lower per nozzle flow rates and at smaller nozzle cross sections it was difficult to illuminate and observe the finer mist, hence the photographs presented here are at the high end of the flow rate range that was studied.

It can be observed in Figure 3 that the Taylor cones of the nozzles remained parallel with a remarkably small deviation at either end. Further, this deviation angle was found to increase with the liquid flow rate.

It is worth emphasizing that our emitter arrays allow for the stable operation at extremely high flow rates before eventually suffering the post nozzle coalescence often reported with alternative designs from others at much lower flow rates. The arrays with the larger 10 × 10 μm nozzles can be operated without backpressure limitations at flow rates that far exceed the normal nanoflow regime, while the individual nozzles still experience a flow rate well within or even on the low end of this range. For example, in Figure 4c, 15 μl/min was consumed with a low per nozzle flow rate of 300 nL/min. Potential applications are envisioned of the multinozzle emitter arrays for assays at microspray flow rates with reduced ion suppression.

The distance between the emitter array chip and the mass spectrometer inlet as well as the spray voltage were optimized in each experiment. It was found that compared to an uncoated pulled tip emitter (operated typically at 1.5 kV), a higher voltage was needed both for the onset of Taylor cones, as well as for optimal signal, with optimized values between 3.3 and 3.6 kV. The voltage needed to be increased up to the breakdown limit, a likely indication that for those number of emitters in the array, and under those flow conditions, the local electric field is close to being sufficient. A possible explanation would be the interference effects by the electric fields formed by neighboring emitters [5], and the current design perhaps could benefit from the inclusion of an extraction electrode to enhance the shielding between neighboring nozzles.

Optimum signal was found for those emitter arrays with overall widths that were not exceeding that of the slotted inlet of the mass spectrometer (i.e. 2.0 mm).

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This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

PO64083-EN 0614S

FIGURE 4. Total Ion Chromatograms (TIC) showing the stability of the signal in top panels (a)–(c) and, in bottom panels (a)–(c), the time integrated mass resolved spectra (Q1MS mode at 0.7 Da resolution) of Hemoglobin A (1.0 pmol/μL infusion) employing multinozzle emitter chips of 10 × 10 μm each: (a) 25-nozzle array at 3.1 kV at 600 nL/min (24 nL/min per nozzle); (b) 50-nozzle array at 3.6 kV at 600 nL/min (12 nL/min per nozzle); (c) 50-nozzle array at 3.4 kV at 2 μL/min (40 nL/min per nozzle). The base peak intensity as a function of total liquid flow rate is depicted for several emitter arrays (d).

Infusion at Nano and Micro Flow Rates The emitter array was found to produce short and long term stable peak intensities in the mass spectrometer (Figure 4).

It can also be observed in Figures 4a – 4c that the charge state distribution was not dependent number of nozzles or liquid flow rate. Additional studies (not shown) also established that it was independent of other device design parameters, and similar to that of a standard nanospray emitter.

Comparing the signal intensity for a fixed total liquid flow rate between emitter arrays with different number of nozzles with the same 10 × 10 μm cross section (Figure 4d), it can be seen that the emitter arrays show increased signal relative to a standard single uncoated pulled tip nanospray emitter (10 μm diameter).

The advantages provided by the photolithographic reproduction on chip-to-chip reproducibility can be judged by the similarity of signal at various flow rates of three 25-nozzle emitter arrays in Figure 4d.

Nanoflow LC-MS-MS Experiments A nanoflow chromatography SRM experiment was performed for the 25-nozzle (10 × 10 μm) emitter array by targeting six constituents of a mixture of BSA and Enolase in an E. Coli digest: TFAEALR (m/z 404.22), AEFVEVTK (m/z 461.75), IGSEVYHNLK (m/z 580.31), LVNELTEFAK (m/z 582.32), NVNDVIAPAFVK (m/z 643.86), and HLVDEPQNLIK (m/z 653.36). We found that the average peak intensities showed good reproducibility between replicate injections (Figure 5). At the concentrations studied this assay is not signal to noise limited and should well represent the number of ions generated.

The slight peak tailing observed in the chromatograms is indicative of post column dead volume, and we identified places to improve the interface to the LC. We are currently optimizing the connections between the multinozzle emitter arrays and LC column to minimize this dead volume.

Further experimentation is underway to compare emitter arrays with different number of nozzles with varying cross sections. For example, for total flow of 300 nL/min, the flow through one of the nozzles in a 50 emitter array is only 6 nL/min, and a 10 × 10 μm nozzle cross section geometry might be less than optimal. Further experiments with a smaller nozzle for the emitter arrays with larger number of nozzles are required.

Parameter Values Number of nozzles 1, 5, 10, 20, 40, 80 Total array width 1.0, 1.75, 2.0, 3.5 mm Nozzle cross section 3 × 3, 5 × 5, 10 × 10 μm Protrusion of nozzle above wafer ~100 μm Inter nozzle spacing 30, 35, 40, 60, 80, 100, 150, 200 μm

TABLE 1. Design parameters of the silicon-chip-based multinozzle emitter arrays. FIGURE 1. Cross section of the monolithic multinozzle emitter array chip. The chip was produced using standard silicon microchip fabrication techniques. Two wafers were bonded together to form an internal channel. In published experiments, this channel could be packed with different kinds (C4 and C18) of beads for on-chip LC separation.

FIGURE 3. Optical microscope images of separate Taylor cones formed by (a) 10-nozzle emitter array (10 × 10 μm nozzles) at a spray voltage of 2.8 kV and 3 μL/min infusion of Angiotensin I, i.e. a flow rate through each nozzle of 300 nL/min; (b) 25-nozzle emitter array (10 × 10 μm nozzles) at 3.5 kV spraying 2 μL/min of Hemoglobin A, i.e. a flow rate through each nozzle of 80 nL/min; (c) 50-nozzle emitter array (10 × 10 μm nozzles) at 4.2 kV spraying 15 μL/min of “cal-mix”, i.e. a flow rate through each nozzle of 300 nL/min; (d) 50-nozzle emitter array (3 × 3 μm nozzles) at 3.8 kV spraying 5 μL/min of acetonitrile (0.1% Formic Acid): 100 nL/min per nozzle. The area represented in the micrographs measures 3.0 × 2.0 mm.

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FIGURE 2. Scanning electron microscope images of sharpened-end multinozzle emitters: (a) 1-nozzle emitter with a cross section of 10 × 10 μm. (b) 20-nozzle emitter with a cross section of 5 x 5 μm. (c) 40-nozzle emitter with a cross section of 2 × 2.5 μm. The zoom-out and close-up views of each emitter are shown in the subpanels (i) and (ii), respectively. Photographs of (d) the multinozzle emitter mounted in a prototype holder, and (e) the holder mounted on the Nanospray Flex NG ion source in front of the slotted ion transfer tube inlet to the mass spectrometer.

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FIGURE 5. Total Ion Chromatogram (TIC) showing the elution of six constituents from a mixture of BSA and Enolase at a flow rate of 300 nL/min. The bar charts on the right represent the peak area for three replicate on column injections of 10 femtomole using a 25-nozzle emitter array of 10 × 10 μm nozzles at 3.3 kV.

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